For tumor imaging, none of the reported PET radiolabeled folates reported to date has made the complete bench-to-bedside journey except [18F]AzaFol, which made it to patients with metastatic ovarian and lung cancers in a multicenter first-in-human trial. In the near future, however, we expect more clinical trials with folate-based PET radiopharmaceuticals given the increasing clinical interest in imaging and the treatment of FR-related malignancies.The folate receptor-α (FR-α) is overexpressed in many epithelial cancers, including ovary, uterus, kidneys, breast, lung, colon and prostate carcinomas, but shows limited expression in normal tissues such as kidneys, salivary glands, choroid plexus and placenta. FR-α has therefore emerged as a promising target for the delivery of therapeutic and imaging agents to FR-positive tumors. A series of folate-based PET (positron emission tomography) radiopharmaceuticals have been developed for the selective targeting of FR-positive malignancies. This review provides an overview on the research progress made so far regarding the design, radiosynthesis and the utility of the folate-derived PET radioconjugates for targeting FR-positive tumors. For the most part, results from folate radioconjugates labeled with fluorine-18 (t S- and R-stereoisomers, however, only the S-isomer is biologically active and present in mammals Ga-radiolabeled analogue [66Ga]Ga-labeled folate derivative for targeting FR-positive tumors using KB tumor-bearing was subsequently investigated. KB cell line is a cervical cancer cell line that expresses the FR at high levels and is therefore used as the “gold standard” for FR-targeting in in vitro and in vivo studies Ga-deferoxamine-folate revealed a heterogeneous tumor uptake, which was blocked by co-injection of folic acid, indicating specific binding to the tumor. A shortcoming of [66Ga]Ga-deferoxamine-folate, however, was the high kidney and abdominal uptake in addition to the poor PET image qualities resulting from the unfavorable physical properties of 66Ga. 66Ga has a maximum positron energy of 4.15 MeV. For comparison, the most widely used positron emitter, 18F, has a maximum positron energy of 0.63 MeV, which is factor 6.5-fold less. In 2005, Rossin et al. fluorobenzylamine-folate -catalyzed azide-alkyne cycloaddition in high radiochemical yields of 25–35% and high molar activities of 160 ± 70 GBq/µmol. The binding affinity of the γ-folate conjugate was determined to be 18.0 ± 7.1 nM, however, a tumor uptake of only 3.13 ± 0.83% (IA/g) at 45 min p.i. was found in KB tumor-bearing mice. High uptake of 2 was observed in the abdominal region and the bile due to the high lipophilicity of the radiotracer caused by the fluorobutyl moiety. The in vivo results of both tracers 1 and 2 indicated that high lipophilicity of folate conjugates results in a high uptake in the gastrointestinal tract and thus, an unfavorable in vivo distribution profile. Based on these observations, Ametamey and co-workers [18F]fluoro-deoxyglucose folate  was also evident. Although the kidney is a FR-positive organ, the high retention observed could not only be attributed to binding to FR in the kidneys but also to unfavorable pharmacokinetics. Previous studies have demonstrated that the incorporation of an albumin-binding entity into radioconjugates resulted in reduced kidney uptake due to increased circulation time in the blood fluoropyridinecarbohydrazide-folate (6) for targeting the FR, however, in this report no biological data was provided. A few years later, the same group published fluorine-18 labeled pyridinecarbohydrazide-methotrexate conjugates as well as an [18F]FDG-folate derivative and reported biological data of all the derivatives [18F]oligoethyleneglycol (OEG)-folate and a 18F-labeled folate-pHPMA conjugate reported by Schieferstein et al. [18F]fluoro-polyethylene glycol (PEG)-folate  value of 1 nM. In vivo PET imaging and ex vivo biodistribution studies demonstrated high and specific binding to FR-positive tumors. However, the liver uptake of 8 was not optimal. To circumvent this shortcoming, the same group later introduced a PEG12 unit instead of PEG1 into [18F]folate-NOTA-AlF in order to increase its hydrophilicity [18F]folate-PEG-NOTA-AlF was achieved using the procedure reported for the non-PEGylated radiolabeled folate. In PET imaging and ex vivo biodistribution studies, a two-fold less liver uptake was observed when compared to 8, suggesting that the increased hydrophilicity caused an improvement in the pharmacokinetic behavior of the PEGylated folate derivative DBCO- and [18F]Ala-folate. In biodistribution and PET imaging studies, both tracers showed low tumor uptake of only 0.5–1.6% IA/g, however, high kidney and abdominal uptake was observed, which could be explained by the increased lipophilicity of the tracers.A few years ago in 2016, Chen et al.  reportedA-AlF 8, . The radhilicity . The radrivative . In 2018rivative  publishe68Ga (maximum energy of emitted β+ particle (β+max) = 1.90 MeV, t1/2 = 67.7 min), 64Cu , 152Tb (average energy of emitted β+ particle (β+ave) = 1.08 MeV, t1/2 = 17.5 h), 44Sc , 55Co  and 89Zr  have been employed more recently for the radiosynthesis of folate conjugates. Prominent among them is 68Ga given its easy accessibility via the 68Ge/68Ga generator system and a broad application in the clinic. In 2011, Fani et al. Tc-EC20. Choi et al. [68Ga]Ga-HBED-CC-EDBE-folate, which showed no favorable in vivo characteristics.Besides fluorine-18, an increasing number of positron emitting radiometals such as i et al.  publisheolate 9,  and 5,8-m et al.  reportedm et al. . By targz et al.  publishel trials ,80,81,82i et al.  prepared68Ga, a small number of publications are available on other positron emitting radiometals including 152Tb, 44Sc, 64Cu, 55Co and 89Zr, which are currently not widely used due to their restricted availability but are under investigation for potential consideration for future application. In 2012, Müller et al. Tb-cm09  and click-[18F]fluoroethyl-folates (5) was found for [18F]FFA. Abdominal, kidney, liver and intestine uptake of [18F]FFA was evident in the PET images.The first folic acid derivative obtained via the integrated approach was reported in 2010 by Ametamey and co-workers . The 2′-al yield . Competi18F-labeling via nucleophilic aromatic substitution can be accomplished by the use of ortho- or para-substituted heteroaromatic rings such as pyridines fluorofolic acid  value of 0.8 ± 0.2 nM to FR. This binding affinity value is similar to that of native folic acid (1.1 ± 0.4 nM) and suggests that the replacement of carbon atom with a nitrogen atom in the phenyl moiety of the folic acid has no detrimental effects on the in vitro binding affinity to FR. Indeed, and as anticipated, the radiosynthesis of 16 could be accomplished in a decay corrected radiochemical yield of 3–9% over two steps, which was higher compared to 15. In addition, 16 showed a higher tumor uptake of 12.6 ± 1.77% IA/g at 90 min p.i. compared to 15. Uptake of [18F]AzaFol in FR-positive kidney and the liver was comparable to the uptake values of [18F]FFA. From these radiochemical and biological results, it can be concluded that compared to [18F]FFA, [18F]AzaFol is more suitable for transferring to an automated synthesis module for possible translation to the clinic.Facilitated yridines . As such-workers  designed11C-labeled compound  to FR-α. Comparable high tumor uptake values (8–11% IA/g) were found for the reduced folates compared to the FA analogues despite the lower binding affinity of the 5-MTHF derivatives. Interestingly, the R- and S-isomers exhibited different absolute uptake values in various organs including the kidneys and the liver, however, no significant changes in the uptake were found for both the α- and γ-conjugated diastereoisomers. This was explained by the different pharmacokinetics of the R- and S-isomers, which show different affinities to transport systems such as the PCFT. The authors concluded that further experiments needed to be done in order to confirm this pharmacokinetic behavior and also experiments to investigate their selectivity for FR-α over FR-β. Nevertheless, this study demonstrated that radiolabeled reduced folates represent a promising alternative to FA for targeting FR-positive cancer tissues.Until recently, the development of folate radiopharmaceuticals mainly focused on folic acid-based derivatives, and no alternative folate-derived structure was considered. This may be explained by the higher synthetic accessibility, the greater stability and the higher binding affinity towards FR of the oxidized form of FA derivatives compared to reduced folates. Importantly, reduced folates exhibit an interesting characteristic by showing subtype selectivity for the FR-α over FR-β. The FR-β isoform is overexpressed mainly on activated macrophages involved in inflammations. Selective tumor over inflammation imaging would therefore be possible with reduced folate radiopharmaceuticals. A selection of radiolabeled reduced folate derivatives reported in the literature is shown in MTHF 18, , a dimet in 2018 . Four isives 19,  were synR- and 6S-3′-aza-2′-[18F]fluoro-5-MTHF derivatives . An explanation for this low radiochemical yield is the lower chemical stability of 5-MTHF compared to FA. Both 5-MTHF isomers exhibited lower and similar binding affinities to the FR-α (27 and 24 nM) compared to the oxidized form [18F]AzaFol 16 (1.4 nM). Biodistribution studies, however, revealed a high unprecedented tumor uptake of over 32% IA/g for both 6R- and 6S-20 at 3 h p.i., whereas for the corresponding oxidized FA derivative 16 only 15% IA/g was found in the KB tumors. Similar to the findings of the R- and S-click-[18F]fluoroethyl-5-MTHF isomers (19), different radioactivity uptake of the 6R-20 and 6S-20 in the kidneys, liver, salivary glands and spleen was also observed. The authors suggested that different transport mechanisms of the three aza-folates  by the folate transporting systems might be a reason for the observed different biological behavior. In general, the reduced radiolabeled folates outperformed the corresponding oxidized form in terms of tumor visualization in PET images  facility in order to guarantee a sufficient amount of product for the patients.Finding a promising lead candidate for further investigation leading to clinical studies requires profound knowledge and a line of research and development in the fields of target identification, organic chemistry, radiochemistry, pharmacology and medical needs . Key par18F-labeled folate derivatives are obtained in low to moderate radiochemical yields and HPLC (high-performance liquid chromatography) purification of the final product is required to achieve a radiochemical purity of >95%. In contrast, radiolabeling of folate conjugates with radiometals normally affords the final product in high radiochemical yields and purity after extensive optimization of the labeling conditions.Based on the research performed during the last decades, it became evident that the (radio)chemistry behind folate derivatives is challenging and not straightforward especially in the case of reduced folates. Reduced folates have decreased chemical stability and are more sensitive to oxidation than the fully oxidized FA, and therefore induce additional challenges during their radiosynthesis. Generally, For preclinical investigations (in vitro and in vivo) of radiolabeled folate derivatives, cervical FR-expressing KB tumor cell lines and xenografts have evolved as the standard model. Nonetheless, tumor models involving other cell lines such as IGROV-1 (ovarian), SKOV-3.ip (metastases-like tumors) or SKBR3 (breast) can be employed . These cR-20 and 6S-20, which showed an IC50 value of around 25 nM and exhibited a tumor uptake of >32% IA/g at 3 h p.i., suggest that tan IC50 value as high as 30 nM may be sufficient for the in vivo imaging of the FR. Second, the lipophilicity  of folate derivatives seems to be an important parameter since data in the literature suggests that folate derivatives with logD values less than −3 generally show more favorable biological characteristics compared to radiotracers with logD values greater than −3. Third, the size (molecular weight) of the folate derivative seems to be of less importance for a promising biological performance as favorable results were obtained with compounds ranging from ~500–2000 g/mol. Finally, for derivatives using the pendant approach, the site of modification at the glutamate moiety should be considered carefully especially for the oxidized forms since in vivo biological characteristics may differ for the different regioisomers.Several cut off criteria exist for the selection of radiolabeled folates for clinical translation. First, high binding affinity to the FR is a necessary criterion. The results obtained for the derivatives 668Ga and 18F and potentially also 44Sc with a half-lives of more than one hour are potentially suitable for imaging FR-positive tumors.Based on preclinical results obtained so far from the many different radiolabeled folate derivatives reported, it has become clear that high tumor uptake and good visualization of FR-expressing tumor tissue in mice can be achieved within the first hours since tumor uptake of so far reported radiolabeled folates did not considerably increase beyond a period of 2–3 h. For this reason, radionuclides such as Once a candidate with promising attributes from the preclinical study has been identified, a proof-of-concept study in humans can be performed. Several important considerations for translating a folate radiopharmaceutical for human studies have to be taken into account and includes the Guidelines of GMP. The availability of a dedicated GMP-laboratory for the radiosynthesis and formulation of the radiotracer is therefore essential. The GMP facility ensures good quality of the final product complying with the current GMP guidelines. To support the submission of dossiers to the relevant health authorities for approval, it is mandatory that preclinical toxicological studies with the non-radioactive substance are performed prior to the first-in-human trial in order to investigate possible toxic effects of the radioligand to be evaluated. However, the risk for toxicological effects of PET imaging probes is generally considered as low as the injected amount is below the therapeutic dose. Finally, for the radiopharmaceutical to be used clinically, local authorities must give the authorization and approval for its use.18F]AzaFol (16) is currently being evaluated in a multicenter first-in-human trial  investigating the biodistribution and FR-specific tumor detection in patients with FR-negative and FR-positive metastatic ovarian and lung cancers AzaFol revealed the highest absorbed doses to the liver, the kidneys, the urinary bladder, and the spleen and the doses were 51.9, 45.8, 39.1 and 35.4 µGy/MBq, respectively. A mean dose of 18.0 ± 2.6 µSv/MBq corresponding to an effective dose of 5.9 ± 0.8 mSv (range 5.4–7.2 mSv), which is comparable to [18F]FDG, was found. Completion and publication of the complete set of the results of this promising first-in-human study with [18F]AzaFol is eagerly awaited. To the best of our knowledge, this is the only clinical study with a fluorine-18 labeled PET folate radiopharmaceutical reported so far in patients with metastatic ovarian and lung cancers. Since in the meantime the corresponding reduced folate derivatives, 6R-20 and 6S-20, in preclinical studies have shown improved performance when compared to [18F]AzaFol, it might be of big interest to also translate these two reduced folate radiotracers to the clinic. The other radiolabeled folate, which is in phase 1 clinical trial not for tumor imaging but rather for differentiating chronic obstructive pulmonary disease (COPD) patients from control subjects, is [68Ga]Ga-EC2115 from Endocyte [To the best of our knowledge, only two PET radiolabeled folates have so far made it to the clinic. [ cancers . Althougl et al. . The PETn et al. . The resEndocyte .FR is a promising target for the detection of FR-positive tumor tissues. As such, a series of chemically different radiolabeled folate derivatives have been designed and evaluated for their utility to target the FR in preclinical settings. The vast majority of the synthesized and biologically evaluated radiolabeled folates did not make the complete bench-to-bedside journey as most of them did not fulfill the stringent requirements for clinical translation. Among other things, the radiolabeled folate should be synthetically easily accessible and radiochemically high yielding; the radiometal for chelation should be easily accessible, affordable and available in sufficient amounts. In addition, the biological characteristics of the radiolabeled folate should be exceptional, i.e., high and specific tumor uptake with a high tumor-to-off-target ratio. If one or more of these prerequisites are not fulfilled, the complex and expensive clinical translation may not be considered. For tumor imaging, only one folate PET tracer labeled with fluorine-18 has recently made the finish line to the clinic. The hope is that more folate-based radiopharmaceuticals with improved selectivity for FR-α over the FR-β isoform, which is overexpressed mainly on activated macrophages involved in inflammation, will be developed. The development of FR-α targeting radiolabeled folates will help to reduce the risk of false- positive results.Finally, the supporting involvement of industrial companies and hospitals in the process of clinical translation of promising candidates is indispensable as major resources and financial support are highly needed for this costly process. Therefore, a close collaboration between universities, companies and hospitals is of major importance to bring folate-based radiopharmaceuticals from bench to the bedside, thereby bringing potential benefits to patients suffering from FR-related diseases, and especially cancers.


Background: The clinical and prognostic value of programmed death-ligand 1, PD-L1, in glioblastoma remains controversial. The present study aimed to identify the expression of PD-L1 for its prognostic value in glioblastoma.Methods: A comprehensive literature search was performed using the PubMed and CNKI databases. The overall survival (OS) and disease-free survival (DFS) of GBM was analyzed based on Hazard ratios (HRs) and 95% confidence intervals (CIs). Furthermore, Odds ratios (ORs) and 95% CIs were summarized for clinicopathological parameters. The statistical analysis was using RevMan 5.3 software.Results: The meta-analysis was performed by using total nine studies including 806 patients who had glioblastoma. The pooled results indicated that PD-L1 expression in tumor tissues was significantly related to a poor OS  with heterogeneity (I2 = 51%). In subgroup analyses, PD-L1 positivity was significantly associated with a worse OS for patients of American and Asian regions, but not for those of European regions. Moreover, PD-L1 expression implied a trend toward the mutation status of the IDH1 gene [coding the Isocitrate Dehydrogenase (NADP(+))-1 protein] . However, the prediction overall survival (OS) of the patients showed that PD-L1 expression was independent from other clinicopathological features, such as gender and age.Conclusions: Our analyses indicated that high expression of PD-L1 in glioblastoma tumor tissues is associated with poor survival of patients, and PD-L1 may act as a prognostic predictor and an effective therapeutic target for glioblastoma. Glioblastoma represents the most commonly seen primary malignant brain tumor in adults, characterized by high aggressive behavior and high recurrence rate . MultimoDifferently from many other tumors, molecularly targeted therapies for glioblastoma produced very limited advances in prolonging life expectancies of the patients, reasons at least partly attributable to the poor penetration of the Blood Brain Barrier (BBB) by therapeutic agents or by rapidly developing drug resistance , 6. In tTumor cells can display immune evasion to weaken antitumor immunity by activating the so-called immune Checkpoint molecules (ICs) . ProgramThe implementation of this systematic review and meta-analysis followed the guideline of PRISM, the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. We systematically reviewed the literature published in the PubMed and CNKI  databases (dated to July 2019). The following key words were adopted:  AND (“CD 274” OR “PD-L1” OR “Programmed Cell Death 1 Ligand 1”) without restrictions on languages, regions and publication types.The study adopted the following inclusion criteria: (1) All patients were diagnosed with glioblastoma by histological examination; (2) Hazard ratio (HR) and 95% confidence intervals (CIs) could be available; or the association between PD-L1 and overall survival (OS) or disease-free survival (DFS) with sufficient data were provided.The study excluded the following: (1) conference abstracts, case reports, reviews, basic research, clinical trials; (2) studies missing available data.Two investigators independently reviewed potentially relevant studies in order to minimize bias. A third reviewer was brought in when there were disagreements. We extracted the following data from the included studies: authors, name of the journal, year of publication and ethnicity, number of enrolled patients, tumor histology, PD-L1 expression level, cut off value, detection area, detection methods, and follow-up.If only survival curves were available, the data could be extracted from the Kaplan Meier curves. The quality of each retrieved article was assessed independently by two assessors according to the Newcastle-Ottawa Quality Assessment Scale (NOS). A total score of 0–9 was assigned to each included study, and studies with a NOS score≥5 were considered to be of high quality .2 statistic. We used a random-effects model to pool the data when evidence suggested significant heterogeneity (I2>50% or P < 0.1), while a fixed-effects model was conducted otherwise. Subgroup analyses and sensitivity analyses were attempted to explain the origin of heterogeneities. The potential publication bias was estimated by the Begg's and Egger's tests with significance of P < 0.05. Review Manager Version 5.3  and STATA 15 were statistical packages used in the study.The association between PD-L1 expression with OS and DFS of patients with glioblastoma was evaluated according to the HR and 95%CI. Statistical heterogeneity among studies was quantified with the Cochran's Q test and the IA total of 201 potentially relevant records were obtained according to the search strategy mentioned above. One hundred and eighty-eight studies were rejected after screening the titles and abstracts. Thirteen studies were included for further evaluation, of which 4 articles without eligible survival data were excluded. Finally, nine studies with 806 patients fulfilled the criteria and entered the meta-analysis. The selection flowchart and the baseline information of the studies are, respectively, displayed in n = 806). Significant heterogeneity existed amongst studies included in the analyses . Pooled result by a random-effects model revealed a significantly inverse correlation between PD-L1 overexpression and OS of patients with glioblastoma  (Nine studies presented OS data (= 0.003) .n = 299) focused on DFS and no heterogeneity was existed amongst the studies . However, pooled analysis by fixed model did not reveal any significant link between PD-L1 and DFS of patients .As shown in P = 0.78).Two studies, consisting of 246 patients, assessed the correlation between age and PD-L1 expression. As shown in P = 0.44; The dependability between PD-L1 expression and gender was assessed in three studies involving 300 patients. Eighty-five (52.47%) of 162 male patients and 64 (46.38%) of 138 female patients were PD-L1 overexpression. The results indicated that PD-L1 overexpression had no significant association with gender  and the American regions , but not in patients from the European studies   .IDH1 mutation (namely IDH1-wild type vs. with IDH1 mutation) and PD-L1. Of the 183 tumors which displayed IDH1-wild type, 67 (36.61%) were PD-L1 positive. Of the 26 tumors which had IDH1-mutantion, one (3.85%) was PD-L1 positive expression. The pooled OR indicated that PD-L1 positivity was closely related to IDH1 status   .P < 0.00001, I2 = 91%).In a subgroup analysis using a random effects model, heterogeneity was revealed in relation to PD-L1 and ethnicity of the patients   .P = 0.003) in glioblastoma patients after surgery; however, there were insufficient evidence to suggest that PD-L1 was related to DFS . These results suggested that PD-L1 positive expression might be a negative prognostic factor in glioblastomas.PD-L1 is a coinhibitory ligand expressed in many types of tumor cells. It has been indicated that the binding of PD-L1 to its receptor PD-1 induces T cell dysfunction and apoptosis which plays a crucial role in tumor immune evasion. Gliomas have been recognized as an immunosuppressive tumor. The current understanding to glioma-mediated immunosuppression have generated increasing interest in the correlations between PD-L1 expression and survival for gliomas, particularly glioblastomas. However, the published results about glioblastomas remain controversial. In 2013 Liu et al. first reported that the expression of PD-L1 in seventeen patients with glioblastoma is a possible indicator for poor clinical outcome . Using lP-values for OS were shown in different ethnic groups: PD-L1 overexpression was significantly associated with poor OS for patients from Asia and America, while no significant association for the survival of patients from Europe survival, which suggested that racial differences may be a potential origin of heterogeneity in glioblastoma. This “ethnic biasedness” of PD-L1 has been observed in several clinical studies for patients with certain other types of solid tumors. KEYNOTE-181, a phase 3 trial of Pembrolizumab (P) vs. chemotherapy  in patients with esophageal squamous cell carcinoma, showed that P was superior to chemotherapy for OS in patients with PD-L1 positive expression (CPS≥10) in the global cohort, especially in the Chinese subgroup  has been considered as one of the important molecular biomarkers that has diagnostic, prognostic and predictive application (IDH1 (an isoform of IDH) generally have a significantly better prognosis compared with IDH1-wildtype glioblastomas  classification of diffuse gliomas, Isocitrate dehydrogenase (lication –42. Studlastomas –45. Morel status , 46, 47.of PD-L1 , 49. So Recent research has showed that tumor cells in gliomas can regulate PD-L1 expression via two major mechanisms to mediate immune evasion, “adaptive resistance” mechanism and “innate resistance” mechanism . The forIt has been indicated that PD-L1 may be a valuable therapeutic target in cancer immunotherapy , 61. RecIDH1 status group, had small sample sizes. Thirdly, the investigation about the correlations between PD-L1 and clinical features, including as tumor size, tumor site and surgical approach are not performed due to the deficiency of related original information.So far as we are aware, the present study is the first meta-analysis to systematically estimate the correlation between PD-L1 and clinical outcomes and clinicopathological factors in glioblastoma. While some limitations need attention. Firstly, different analysis strategy of IHC and inconsistent cut-off values of PD-L1 expression may lead to heterogeneity between studies. Thus, a standardized approach for protein expression should be set up to improve consistency and veracity in the measurement of PD-L1 for future studies. Secondly, some subgroups, such as the In conclusion, our meta-analysis shows that high PD-L1 expression is strongly correlated with unfavorable prognosis for GBM patients. PD-L1 may present itself as a valuable target for immunotherapy in clinical practice. Additional high-quality, larger-scale prospective studies are needed to provide validate the potential value of PD-L1 for the prognosis and treatment of GBM patients in the future.The datasets generated for this study are available on request to the corresponding author.CH and GC designed this study, searched the literature, screened identified studies and extracted data, and wrote the manuscript. Disagreements were resolved by discussion with WJ and WL. HZhao, FC, YL, SL, HZhang, YZ, and JC assisted in the literature search and screening. WJ and WL assisted in the designing of the study and the data analyses, and supervised the study. All authors have read and approved the final version of this manuscript.GC was employed by the company Beijing Qinglian Biotech, Co., Ltd., Beijing, China. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tracer accumulation was quantified by PET and tumor volumes measured with MRI (n = 42). Moreover, gamma-counter analysis of radiotracer accumulation was done ex-vivo. A three- to five-fold higher ligand accumulation in the PSMA-positive tumors compared to the PSMA-negative tumors was demonstrated. This proof-of-principle study shows the general feasibility of the HET-CAM xenograft model for target-specific imaging with PET and MRI. The ultimate value for characterization of novel target-specific radioligands now has to be validated in comparison to mouse xenograft experiments.The validation of novel target-specific radioligands requires animal experiments mostly using mice with xenografts. A pre-selection based on a simpler in vivo model would allow to reduce the number of animal experiments, in accordance with the 3Rs principles . In this respect, the chick embryo or hen’s egg test–chorioallantoic membrane (HET-CAM) model is of special interest, as it is not considered an animal until day 17. Thus, we evaluated the feasibility of quantitative analysis of target-specific radiotracer accumulation in xenografts using the HET-CAM model and combined positron emission tomography (PET) and magnetic resonance imaging (MRI). For proof-of-principle we used established prostate-specific membrane antigen (PSMA)-positive and PSMA-negative prostate cancer xenografts and the clinically widely used PSMA-specific PET-tracer [ The development of target-specific radiolabeled probes like on the basis of small molecules, peptides, antibodies, or nanoparticles, always requires information on biodistribution and in particular on the level of specific binding of the novel probe to the target in the in vivo situation, which means specific radiotracer accumulation in the target-expressing region of interest ,2,3,4,5.In this respect, the hen’s egg test-chorioallantoic membrane (HET-CAM) model is a promising alternative for animal experiments, as in many countries, chick embryos are not considered live animals before embryo development day (EDD) 17 or hatching. Thus, in contrast to animal studies, the HET-CAM model does not rise legal authorization issues if the embryos are sacrificed accordingly. Considering the fact that there is some dissent in the available literature concerning the point at which the chick embryo starts to feel pain, it is recommended to initiate the experiments as early as possible and to use methods with minimal burden ,12,13,14Recently, several studies have been published about tumor imaging in the HET-CAM model, focusing on MRI compounds  or thera68Ga]Ga-PSMA-11  , [LNCaP / PC-3]  .We also performed dynamic measurements of radiotracer accumulation over time, however, as measurements normally started 10 min after injection, no perfusion peak can be observed in the time–activity curves and thus quantitative kinetic modelling was not performed . In a quIn the body of the embryo, the activity was heterogeneously distributed with highest accumulation in the heart region  %IA all PSMA+, (0.11 ± 0.11) %IA all PSMA− PC-3; p < 0.0001). When separately analyzing the subgroup of LNCaP C4-2  %IA; median: 0.24 %IA) and excluding the five tumors with more intense spillover and thus potentially impaired data quality, the accumulation still was significantly higher compared to PC-3 (p < 0.001). Even in the five experiments with a more intense spillover, the activity ratios still were substantially higher in the PSMA+ tumors compared to the PSMA-negative tumors . Tracer accumulation was also substantially higher in the small subgroup of LNCaP versus PC-3 ((0.20 ± 0.03) %IA vs. (0.12 ± 0.11) %IA), however did not reach statistical significance because of the low number of experiments . Tracer accumulation was also significantly higher in PSMA-positive tumors compared to background (CAM only) mean 0.34 [LNCaP C4-2 + CAM] n = 5, p = 0.0313. Results of the subgroup evaluations are presented in Absolute radiotracer accumulation evaluated ex vivo by gamma counter measurements showed a significantly higher accumulation in the PSMA-positive tumors in comparison to the PSMA-negative tumors (p < 0.0001). This resulted in high ratios of activity concentration of PSMA-positive versus negative tumors (mean ± SD) of 4.0 ± 3.9 [LNCaP C4-2 vs. PC-3], 4.9 ± 0.8 [LNCaP vs. PC-3], and also versus background with 24.8 ± 48.5 [LNCaP C4-2 vs. CAM]. Furthermore, the activity concentration ratios of the tumor activity in comparison to the total activity in the chick embryo  were determined (p < 0.0001).When using the MR-derived tumor volumes for calculation of the radioactivity concentration in %IA per gram, the values of PSMA-positive tumors were also significantly higher compared to PSMA-negative tumors , however no quantitative analysis was performed because of the missing perfusion phase as measurements could not be started earlier than 10 min. p.i., because the injection is too difficult to be performed in the scanner. Qualitative evaluation did not reveal substantial differences in the patterns of the TACs between PSMA68Ga]Ga-PSMA-11 and other PSMA-specific radioligands have been analyzed by in vivo studies in mice. In + and PSMA− tumors of all ligands listed, the results of our HET-CAM experiments are very comparable and within the same order of magnitude, which further corroborates our data. It is of interest that similar to our results in the HET-CAM model, a minor ligand uptake was also observed for PC-3 tumors in mice. At first glance this is surprising as it was clearly demonstrated in various in vitro studies that the PC-3 cell line is PSMA-negative , validating the CAM in the PC-3 area of the silicone rings. Tumor growth and embryo health were daily monitored by visual inspection. The MR and PET imaging experiments were performed between EDD12 and EDD16. For MRI scans the chick embryos were cooled for 70 to 120 min at 4 °C prior to the measurement start to minimize movement artefacts, according to the protocols of Bain et al. and Zuo et al. [68Ga]Ga-PSMA-11 (1.3 µg/mL) into a chorioallantoic membrane blood vessel. On average an activity of 1.7 ± 1.5 MBq, in median 1.1 MBq was injected. To stop the bleeding after injection a cotton bud was held on the injection side and liquid bandage spray  was pipetted on the dried surface. The complete chick embryo and afterwards also needle, syringe, and cotton buds were measured in an activity meter  to determine the successfully applied radioactivity which was set to 100% injected activity [%IA] for quantification calculation. Events, where blood was slightly distributed across the membrane or near/into the tumor areas were protocolled and considered during the evaluation of the data, while large spillover lead to exclusion from evaluation in four cases. The embryos have been sacrificed after tumor excision.The method we used was similar to already published HET-CAM models ,92,93,94o et al. ,32. The o et al. . For PET68Ga]Ga-PSMA-HBED-CC ([68Ga]Ga-PSMA-11) was produced as recently published Ga-PSMA-11.The eggs were placed in a custom-build holder for enabling PET-MRI registration by specific fiducials filled with 50 µl CuSOThe implemented protocols for MR measurements were based on the publications of Zuo et al. ,31. Imag2, slice thickness = 0.5 mm, no interslice gap, RARE factor = 8 and NSA = 4. The number of slices was adapted to the tumor size and ranged from 15 to 30 slices. The acquisition time for a single high-resolution RARE scan was between 15 and 35 min depending on the number of slices required to cover a sufficient volume of interest, covering both, the tumors and the chick embryos. Tumor volume, location, and structure were assessed by applying a high-resolution T2-weighted multislice rapid acquisition with relaxation enhancement (RARE) sequence. Scan parameters were as follows: TR/TE = 4320/45 ms, matrix size = 650 × 650, in-plane resolution = 77 × 91 μmFor PET imaging a dynamic 60 min scan was performed on a small animal PET scanner . The Focus120 has a high spatial resolution (<1.3 mm) and high sensitivity (about 7%) with a bore size  . These l3, where D was at least 1.5 mm and adapted to the respective tumor size. VOI analysis provided voxel-based mean data for each 5 min time frame.MRI and PET data were fused by fiducial registration using the 3Dslicer software (ver. 4.10.2)  and the +/PSMA−) / tumor volume (PSMA+/PSMA−)] was calculated to provide the value for evaluation of the ligand accumulation. Values > 1 indicate a higher accumulation in the PSMA-positive tumor regions. Using data from the gamma counter and volume determination based on MR images, the accumulated activity in %IA/g was calculated for comparison with literature data. The calculation is based on the definition of 1000 mm3 = 1 g, whereby the density of water as the main component in the cells is approximated.After washing (1 min), the tumors were analyzed with a gamma-counter COBRA II (Perkin Elmer) to accurately quantify the accumulated radioactivity. Quantification of the radioactivity in the tumor was based on these gamma-counter measurements followed by decay correction and calculation of the fraction in comparison to the applied activity . The quotient of the activity ratios and the tumor volume ratios of the paired PSMA-positive and PSMA-negative xenografts [radioactivity in the tumor (PSMAwww.graphpad.com). A p-value < 0.05 was assumed as statistically significant.Wilcoxon matched-pairs signed rank tests were performed using GraphPad Prism  was applied for 60 min followed by treatment using 2% normal serum (blocking serum)  in PBA for 30 min at room temperature. The mouse monoclonal primary antibody YPSMA-1  was applied in a dilution of 1:800 in PBA and the sections were incubated overnight at 4 °C in a wet chamber. After washing in PBS , biotinylated secondary antibody  diluted 1:200 in PBA was applied for 60 min in a wet chamber at room temperature. Sections were rinsed in PBS  and incubated with avidin/biotin-based VECTASTAIN® Elite® ABC Kit (Peroxidase (HRP) working solution  for 30 min at room temperature. Following an additional washing in PBS , Vector® DAB peroxidase substrate  was applied for 10 min at room temperature. The substrate was removed by washing with water  and counterstained using hematoxylin solution modified according to Gill III . After dehydration by ascending alcohol steps (2 min) and toluene , slides were mounted .Tumor excision tissues were fixated in 4% formaldehyde in phosphate-buffered saline (PBS) and paraffin-embedded. Sections were deparaffinized using xylene  and rehydrated by washing in descending alcohol solutions. To reveal the masked epitopes antigen retrieval was obtained by boiling citrate-based antigen unmasking solution . The cooled sections were washed for 1 min using 30% HOur feasibility study suggests that the HET-CAM xenograft model using PET and MR imaging is promising for the initial assessment of specific radiotracer accumulation under in vivo conditions and the results of our study are in line with the reported data in mice xenografts. The model has many advantages compared to the mouse model, e.g., lower costs, less administrative work, a short experimental start-up time, and less maintenance effort. On the other hand, its application is limited by the rather short time window for the experimental procedures, and technical challenges like injecting into the rather small CAM vessels. However, the time window is only of minor importance in the context of the intended approach for the initial analysis of new radioligands and the technical challenges can be overcome with appropriate experience. Now further comparative studies for validation of our data have to be performed. If successfully established, combined PET-MR imaging using the HET-CAM xenograft model would allow for a substantial reduction of animal experiments in line with the 3Rs principles.


Fetal hemoglobin (HbF) is a determining factor for the development of sickle cell anemia. High HbF levels lower the intensity of symptoms of this disease. HbF levels can vary in patients with sickle cell anemia and individuals without the disease. The purpose of this study was to identify the genetic variants in the G gamma-globin gene promoter that can modulate HbF expression in patients with sickle cell anemia and healthy individuals from Colombia. In total, 413 bp of the G gamma-globin gene promoter were sequenced in 60 patients with sickle cell anemia and 113 healthy individuals. The allelic and genotype frequencies of the identified variants were compared between individuals with low and high HbF for both patients and healthy individuals. In total, we identified 15 variants in both groups, only three of which were shared between patients and healthy individuals. In healthy individuals, sites -16 and -309 (rs112479156) exhibited differences in allele frequencies. The mutant allele of -16 lowered the production of HbF, whereas the mutant allele of -309 increased its production. These results reveal the presence of different mechanisms of HbF regulation between patients with sickle cell and healthy individuals. The expression of fetal hemoglobin (HbF), a type of hemoglobin, starts around the fifth week of gestation. HbF consists of two types of globin chains: alpha and gamma. HbF synthesis stops during the perinatal period, at which time it is replaced by adult hemoglobin . In adulBCL11A   were randomly collected from healthy individuals and patients with sickle cell anemia inhabiting the Pacific and Atlantic coasts of Colombia. Of these, 39 were from the San Jerónimo Hospital and ABO Laboratories in Montería (Atlantic coast), 184 were from the Louis Pasteur Laboratory and the Casa del Niño Hospital (Napoleón Franco) in Cartagena (Atlantic coast), 820 were from Buenaventura (Pacific coast), and 93 were from the Valle University Hospital and the Noel Club Children’s Clinic Foundation in Santiago de Cali (Pacific coast). The participants were individuals aged > two years who had not undergone a blood transfusion in the last three months and who had not been treated with hydroxyurea or any other drug that could increase HbF levels. The Ethics Committee of Universidad del Valle approved this research project . The participants or their legal representatives provided signed informed consent before their entry to the survey. Samples were stored at 4 °C in the Genetics Section of the Human Molecular Genetics Laboratory at the Universidad del Valle, Meléndez campus. From the 1136 samples, 60 individuals were diagnosed with sickle cell anemia (SS group), and a random sub-sample (n = 113) of individuals without sickle cell anemia was created (AA group).DNA was extracted from each sample using the salting-out method  were mixed to achieve a final volume of 25 µL. Incubation was performed in a warm water bath at 37 °C for at least 16 h.The genotype in the HbS locus was determined via PCR amplification of a 596-bp fragment of the beta-globin gene and subsequently digested with the HBG2. This sentence should be: The premix was defined as follows: buffer 1X; , 0.98 µM; dNTPs, 0.01 µM; primers, 0.12 µM; Taq DNA polymerase, 1 IU; and DNA, 50 ng. The amplification program was initiated with heating at 92 °C for 5 min, followed by 30 cycles of denaturation at 93 °C for 36 s, annealing at 70 °C for 1 min, and extension at 72 °C for 1.5 min. Subsequently, a final extension was performed at 72 °C for 3 min. Confirmation of the amplification was performed via electrophoresis using a 29:1 polyacrylamide gel at 8%, with a 413-bp band corresponding to the promoter region. The determination of the nucleotide sequence of HBG2 promoter region (413 bp) was performed using Sanger-type sequencing with the BigDye® Terminator v3.1 Cycle Sequencing kit  following the amplification program suggested for this kit. Sequencing was performed using an ABI 3130 unit (Applied Biosystems) that was available at the Human Molecular Genetics Laboratory at the Universidad del Valle.The primers CAGGCCTCACTGGAGCTACAGAC (forward) and ACCTCAGACGTTCCAGAAGCGAGT (reverse) were used to amplify the promoter region of http://CRAN.R-project.org/package=haplo.stats).The sequences were processed and analyzed using Sequencing analysis v. 5.3.1 (Applied Biosystems) and aligned with the SeqScape program v. 2.7 (Applied Biosystems) where the different polymorphisms were identified. The allelic frequency of each single nucleotide polymorphism (SNP) was determined via direct counting in both the AA and SS groups. An estimation of the gametic phases was performed using the Arlequin program v. 3.5  and the The allelic frequencies of the SNPs were compared in individuals with high and low HbF for both the SS and AA groups using the Raymond & Rousset test  with ArlHBG2, we found 15 variants in the entire population sample. The variants were observed at positions -356, -325, -324, -322, -317, -309, -307, -271, -203, -168, -161, -158, -152, -53, and -16. Six of these variants had previously been reported in NCBI: -324 (rs78485026), -317 (rs74929542), -309 (rs112479156), -307 (rs112219751), -271 (rs113622787), and -158 (rs7482144). However, no reports were found for the remaining nine sites. Seven variants were identified in patients with sickle cell anemia, and 11 were observed in healthy individuals. It is important to note that only three of these polymorphisms  were shared between patients and individuals without the disease  and variance homogeneity  of HbF levels were evaluated. ANOVA revealed that individuals homozygous for TT at -309 had lower HbF levels than those with at least one mutated C allele (TC and CC) . For the polymorphism at position -16, individuals with the CC genotype had higher HbF levels than those with the CG or GG genotype  . We idenRegarding LD between the restriction sites, sites -356, -325, -317, -309, -307, and -271 formed a linkage block with high levels of LD in the SS group. Site -158 exhibited intermediate levels of LD. Concerning healthy individuals, a linkage block was formed between sites -325 and -161. However, unlike the case of SS patients, this block has intermediate LD levels between some sites. LD levels were intermediate outside the linkage block, and some sites did not display LD, which could indicate recombination events .HbF is a factor that modulates the severity of sickle cell anemia, and hence, its regulation has been well studied . In our Only two polymorphisms (-309 and -16) displayed differences in allelic frequencies between individuals with high and low HbF levels, although this difference was only observed in individuals without sickle cell anemia. Previous reports of these two variations are scarce. For both cases, noninterference with HbF expression has been reported. In the case of -16, this polymorphism alters a putative CACCC box located between the TATA box and transcription start site. Despite this, -16 did not affect HbF expression in the adult or fetal state in an African American population   that generate a binding site for stage selector proteins that cause hereditary persistence of HbF . These differences in heterozygosity could be the product of an interbreeding process that is more widespread in healthy populations than sickle cell populations.HBG2 promoter. No variants that were associated with HbF expression in patients with sickle cell anemia were found in our population, but two variants (-16 and -309) are associated to the HbF regulatory process in individuals without sickle cell disease. The presence of different groups of polymorphisms could also indicate that there may different mechanisms of HbF regulation between patients with sickle cell anemia and individuals without the disease.In conclusion, chromosomes S and A contain different groups of genetic variants in the


A global, quantitative proteomics/systems-biology analysis of the selective pharmacological inhibition of phosphodiesterase-4D (PDE4D) revealed the differential regulation of pathways associated with neuroplasticity in memory-associated brain regions. Subtype selective inhibitors of PDE4D bind in an allosteric site that differs between mice and humans in a single amino acid . Therefore to study selective inhibition of PDE4D by BPN14770, a subtype selective allosteric inhibitor of PDE4D, we utilized a line of mice in which the PDE4D gene had been humanized by mutating the critical tyrosine to phenylalanine. Relatively low doses of BPN14770 were effective at reversing scopolamine-induced memory and cognitive deficits in humanized PDE4D mice. Inhibition of PDE4D alters the expression of protein kinase A (PKA), Sirt1, Akt, and Bcl-2/Bax which are components of signaling pathways for regulating endocrine response, stress resistance, neuronal autophagy, and apoptosis. Treatment with a series of antagonists, such as H89, sirtinol, and MK-2206, reversed the effect of BPN14770 as shown by behavioral tests and immunoblot analysis. These findings suggest that inhibition of PDE4D enhances signaling through the cAMP-PKA-SIRT1-Akt -Bcl-2/Bax pathway and thereby may provide therapeutic benefit in neurocognitive disorders. Note that affiliations are listed sequentially as per journal style and requests for non-sequential listing will not be applied.Alzheimer’s disease (AD) is characterized by progressive decline in learning and memory with accompanying neuropathological changes. The disease is a major cause of disability and mortality in aged people. Currently, epidemiological investigations suggest that approximately 44 million people worldwide live with cognitive deficits associated with AD . That nuClinical investigation indicates the therapeutic potential for inhibition of phosphodiesterase-4 (PDE4) for neuropsychiatric disorders . HoweverPhosphodiesterase-4D is highly expressed in the cortex and hippocampus , integraIn the present study, quantitative proteomics analysis was used to identify proteins whose expression was altered by the PDE4D inhibitor BPN14770 and the biochemical processes associated with these proteins. This supports the hypothesis that PDE4D modulates the Sirt1-Akt pathway in ways relevant to learning and memory. Scopolamine- impairment reduces Sirt1 and Akt expression in the hippocampus and this is reversed by the selective inhibition of PDE4D by BPN14770. We also show that scopolamine-impairment reduces the ratio of Bcl-2/Bax in hippocampus and that this too is reversed by BPN14770. The cognitive benefit of BPN14770 in the scopolamine-impairment model is blocked by pre-treatment with inhibitors of PKA (H89), Sirt1 (sirtinol), and Akt (MK-2206). This suggests that the PKA-SIRT1-Akt pathway is a potential therapeutic target for BPN14770 when it comes to protecting subjects from memory and cognitive impairment such as occurs in AD.g). The experimental procedures were carried out following the National Institutes of Health Guide for Care and Use of Laboratory Animals , and approved by the Institutional Animal Care and Use Committee at University at Buffalo, The State University of New York.Phosphodiesterase-4D humanized mice (C57BL/6 strain) and littermate (WT) controls were produced by InGenous Targeting Laboratory  under contract to Tetra Discovery Partners, Inc. and bred at the University at Buffalo . They weThe selective PDE4D allosteric inhibitor BPN14770 (CAS # 1606974-33-7) was synthesized as described . BPN1477For the proteomics analysis, male and female humanized PDE4D mice were dosed once daily for 5 days with BPN14770 or vehicle and the brains were harvested 1 h after the last dose. The mice were sacrificed by cervical dislocation, the hippocampus and frontal cortex were harvested, weighed, and then stored in a cryo-freezer set to –80°C.The flow chart for proteomics analysis of humanized PDE4D treated with BPN14770 or vehicle is shown in P < 0.05 as the cut-off values for differential protein quantification  package with fold change >1.2 or <0.83, and fication . Comparification . The STRfication . The genfication . Predictfication . The KEGfication .For the Morris Water Maze (MWM) and Step-Down Passive Avoidance tests, male and female humanized PDE4D mice were treated by oral gavage with BPN14770 or vehicle once daily for 5 days and their behavior was assessed each day 1 h after dosing. Scopolamine was administered by intraperitoneal injection 30 min before administration of BPN14770 or vehicle. For behavioral assessment in the scopolamine-impairment tests, male and female mice were randomly divided into six groups (10 mice per group) as follows: a control group with no treatment, a scopolamine-impaired group treated with vehicle by oral gavage, groups treated with BPN14770 at 0.003 mg/kg (low dose), 0.01 mg/kg (medium dose), and 0.03 mg/kg (high dose), and a positive control group treated with rolipram, a reference PDE4 inhibitor. Mice were trained in the MWM task 1 h after dosing on days 1, 2, 3, and 4 with the hidden platform test performed on day 5 at 24 h after the last training session. Mice were trained on the Step-down Passive Avoidance task on days 3 and 4 and then tested for step down avoidance on day 5. The MWM test was performed immediately after the Step- down Passive Avoidance test on days 3, 4, and 5. Brain tissues for immunoblot analysis were harvested immediately after the MWM hidden platform test on day 5. In addition, three groups of mice were pretreated 30 min before administration of the high dose of BPN14770 or vehicle with the PKA inhibitor H89, the SIRT1 inhibitor sirtinol, or the Akt phosphorylation (pAkt) inhibitor MK-2206 once daily for 5 days. Behavioral testing was conducted as above and the brains were harvested immediately after the MWM test on day 5 and processed as above.The protocol for the Step-Down Passive Avoidance Test followed a recent study with slight modifications . An appaThe procedure for carrying out the MWM test was nearly identical to those followed by previous experiments . A circuAll tissue samples were homogenized in radio-immunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitors and centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was mixed with 2× loading buffer and heated to 95–100°C for 5 min to denature the proteins. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay. Samples (50 μg protein each) were separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) before transferring to polyvinylidene fluoride (PVDF) membranes . The membranes were incubated with 5% bovine serum albumin, a blocking buffer to eliminate effects of non-specific bindings for 1 h at room temperature. Afterward, the blots were incubated with the primary antibodies overnight at 4°C on a shaker: mouse anti-SIRT1 , mouse anti-actin  rabbit anti-Akt , rabbit anti-phospho-Akt , rabbit Bcl-2 , and rabbit anti-Bax . After being washed with tris-buffered saline and tween 20 (TBST), the membranes were incubated with the secondary antibodies on a shaker: goat anti-mouse IgG-HRP (1:3000) goat anti-rabbit IgG-HRP  for 60 min at room temperature. Band signal was visualized by the enhanced chemiluminescent (ECL) kit (Millipore) and detected by a fluorescence scanner. The resulting images of the bands in question were visualized by an Image-Lab software device.t-test. All test results that yielded a P-value less than 0.05 were considered statistically significant.After the images of the blots were collected, their relative protein amounts were quantified by Image J (NIH shareware). The data from the behavioral tests and Immunoblots were analyzed using GraphPad Prism 7.0. Results are expressed as mean ± standard error of the mean (S.E.M.). All data were analyzed statistically by one-way analysis of variance (ANOVA), followed by Dunnett’s P < 0.05 as the cut off value for statistical signficance. For example, multiple tryptic peptides from PDE4A, PDE4B, and PDE4D were identified and their levels were unchanged in cortex or hippocampus by treatment with BPN14770 . The scopolamine-deficit was reversed in the groups treated with BPN14770 at doses of 0.003, 0.01, and 0.03 mg/kg which exhibited a greater tendency to avoid stepping onto the electrified floor of the chamber as compared to the vehicle-treated scopolamine group , P < 0.05).In order to evaluate the effects of BPN14770 in preclinical models of learning and memory disorders, cognitive deficits were induced by treatment with scopolamine 1 h prior to dosing with BPN14770 once daily for 5 days. Mice were evaluated for scopolamine-induced cognitive deficits in the Step-Down Passive Avoidance Model and the MWM. Mice were trained in the Step-Down Passive Avoidance Model on days 3 and 4 of dosing and tested for memory retention on Day 5. The Step-Down Passive Avoidance test was administered immediately prior to the MWM. As shown in P < 0.05), further corroborating the idea that PKA-related signaling interacts withThe preceding proteomics analysis suggested that the benefit of the PDE4D allosteric inhibitor might be through modulation of cAMP-PKA-CREB and Sirt1-Akt1-Bax signaling modules. Therefore, we explored the effect of inhibiting PKA, Sirt1, or Akt1 on the pharmacological response to BPN14770. H89, a PKA inhibitor, administered 30 min prior to BPN14770 each day blocked the benefit of BPN14770 (P < 0.05), PDE4D inhibitors such as BPN14770 . MoreoveP < 0.05; P < 0.001; F = 5.955, P = 0.0006] at doses of 0.01 mg/kg (P < 0.05) and 0.03 mg/kg p.o. (P < 0.01), P’s < 0.01; or P < 0.05, The MWM test was administered 1 hr after dosing with BPN14770 or vehicle over five successive days. On day 1, all groups took similar time to find the platform. However, by days 3 and 4 the scopolamine-treated mice took considerably longer to reach the platform on as compared to their vehicle-treated littermates  = 3.25, P = 0.02] at a dose of 0.03 mg/kg p.o. (P < 0.05). The inhibitors H89, sirtinol, and MK-2206 counteracted this benefit of BPN14770 . However, the decrease in SIRT1 expression in the hippocampal was ameliorated by treatment with BPN14770 at 0.03 mg/kg . The effects of BPN14770 on SIRT1 expression were reversed by pre-treatment with H89 or sirtinol . The reduction in the ratio of pAkt to Akt was reversed by administering BPN14770 at 0.01 and 0.03 mg/kg p.o. . The fact that the ratio was higher for the greater concentrations of the drug implies a dose-dependent therapeutic effect . Furthermore, pretreated either H89, Sirtinol, or MK-2206 prevented the increase in hippocampal p-Akt to Akt ratio bestowed by BPN14770  to Bax ratio is an indicator of the extent of apoptosis in the target tissue. In our study, the proteomics results showed that Bax was up-regulated in the hippocampus, and Bcl2 was down-regulated in the cortex after treatment with BPN14770, which did not meet our prediction . To veriP < 0.01) as shown in F = 14.83, P < 0.0001]. At 0.01 or 0.03 mg/kg doses, the average ratio of Bl-2/Bax was close to that of the vehicle-treated control mice . The PKA inhibitor H89 reversed the BPN14770-induced increase in Bcl-2/Bax ratio  in the PI3K/AKT/mTOR pathway . MammalsSome studies have shown that cells may have an advanced mechanism that allows mTORC1 to immediately respond to the cAMP signal via the dual function of PDE4D . The posIn conclusion, the results of our study demonstrate that subacute treatment with PDE4D inhibitor BPN14770 is effective at alleviating scopolamine-induced memory and cognitive impairment. The underlying mechanism may be involved in the neuroprotective and antiapoptoticeffects of BPN14770 by regulation of the cAMP-PKA-SIRT1-Akt -Bcl-2/Bax pathway.The original contributions presented in the study are included in the article/The experimental procedures were carried out following the National Institutes of Health Guide for Care and Use of Laboratory Animals , and approved by the Institutional Animal Care and Use Committee at University at Buffalo, The State University of New York.YW and VZ performed the experiments. SG, LC, and HZ conducted the bioinformatic analyses and wrote the manuscript involved this part. MM, SS, and JQ were responsible for the quantitative proteomics/systems-biology analysis. MG reviewed and edited the manuscript. YX and JO’D conceived and designed the study. All authors contributed to the article and approved the submitted version.MG is an employee of Tetra Therapeutics which has a financial interest in BPN14770. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hyperhomocysteinemia (hHcy) represents a strong risk factor for atherosclerosis-associated diseases, like stroke, dementia or Alzheimer’s disease. A methionine (Met)-rich diet leads to an elevated level of homocysteine in plasma and might cause pathological alterations across the brain. The hippocampus is being constantly studied for its selective vulnerability linked with neurodegeneration. This study explores metabolic and histo-morphological changes in the rat hippocampus after global ischemia in the hHcy conditions using a combination of proton magnetic resonance spectroscopy and magnetic resonance-volumetry as well as immunohistochemical analysis. After 4 weeks of a Met-enriched diet at a dose of 2 g/kg of animal weight/day, adult male Wistar rats underwent 4-vessel occlusion lasting for 15 min, followed by a reperfusion period varying from 3 to 7 days. Histo-morphological analyses showed that the subsequent ischemia-reperfusion insult (IRI) aggravates the extent of the sole hHcy-induced degeneration of the hippocampal neurons. Decreased volume in the grey matter, extensive changes in the metabolic ratio, deeper alterations in the number and morphology of neurons, astrocytes and their processes were demonstrated in the hippocampus 7 days post-ischemia in the hHcy animals. Our results suggest that the combination of the two risk factors (hHcy and IRI) endorses and exacerbates the rat hippocampal neurodegenerative processes. Methionine (Met) is an essential amino acid present in food, which is regularly consumed within the Western diet ,2,3,4,5.Furthermore, protein intake rich in Met content or dysregulation of Met metabolism within the “Met-homocysteine” cycle can concomitantly lead to the elevation of homocysteine (Hcy) in the circulating plasma. Hcy is a sulphur-containing amino acid produced by metabolic conversion of Met to cysteine. Its elevated level in plasma, hyperhomocysteinemia (hHcy), is one of the known risk factors for cardio- and cerebrovascular disorders. Clinical relevance of elevated total Hcy level in plasma has been proven by many studies on different tissues ,15. ReceIn our previous work on the rat hHcy model induced by a Met-enriched diet , we haveOur experiment was accomplished according to Directive 2010/63/EU of the European Parliament and the Council on the protection of animals used for scientific purposes. Animals used in this experiment were handled in accordance with the directive for Animal Care and Health of the State Veterinary and Food Department of the Slovak Republic  for animal experiments. n = 38) were used in this experiment. Animals were kept under the standard conditions (air-conditioned rooms with a temperature of 22 ± 2°) and 12 h day/night cycle. Water, as well as food, were available ad libitum. Global IRI was evoked by the 4-vessel occlusion (4-VO) model in accordance with Pulsinelli et al.  were obtained in one acquisition using the chemical shift imaging (CSI) method ; 22 × 22 mm2 FOV; TR/TE = 1500/20 ms; 36 averages and 6 kHz acquisition bandwidth. Eddy currents and B0 drift compensations, as well as OVS (outer volume suppression) and VAPOR (variable pulse power and optimized relaxation delays) suppression, were initiated. Linear and second order shims were automatically adjusted with the cuboid shim volume. The average linewidth of water peak was 14.8 ± 1.9 Hz.For identification of magnetic field (B) method . The 2D 2-weighted MRI slices  software on 12 consecutive coronal TI slices . The sel1H MRS data curve fittings were performed by LCModel software . Subsequently, the metabolite levels of total N-acetyl aspartate , myo-Inositol (mIns), choline  and creatine  containing compounds were expressed as following ratios: tNAA/tCr, tCho/tCr, mIns/tCr and mIns/tNAA.During the spectroscopic data assessment, one representative voxel from hippocampus  was sele2-infinity leaving morphology unspecified. A total of 534 fields of view were analysed. The total number of Fluoro-Jade C+ (green fluorescent cytoplasm), NeuN (red fluorescent cytoplasm and processes), GFAP (green fluorescent cytoplasm and processes) was directly counted in the sections of the hippocampal CA1 area of each staining, respectively . The sampling grid size was set up to: 6 × 6 mm for each area. All calculations are expressed as the total number of labelled cells per mm2.Images of the coronal brain sections from the first experimental group were exported in tiff format. Images were evaluated using ImageJ software . Firstly, red, green and blue (RGB) channels were converted to 8 bit grey-scale images. Threshold levels were set from 13 pixels (min) to 255 pixels (max). Particle analysis was finished based upon size restrictions of 0 mmt-test.Data acquired from image analysis of brain sections were analysed using GraphPad Prism software, version 6.01 for Windows . The data were evaluated using one-way and two-way analysis of variance (ANOVA). Statistical analysis for spectroscopic and MRI volumetric data was accomplished using the SPSS software package . The differences in hippocampal volumes and metabolite ratios between animal groups  were analysed by independent-samples 2-tailed n = 5), and reached 11.22 ± 3.86 μmol/l (n = 5). Analysis of plasma level of Hcy in animals has shown that total plasma Hcy level in animals with 28 days of Met diet (Met-C) was significantly higher when compared to the naïve male control Wistar rats , in comparison to both controls (C and Met-C). Interestingly, in the same group with the Met diet (Met-IR-3d), the differences reached 58.33-fold increase , when compared to the naïve ischemized group. To show the extent of neurodegeneration, FJC was used for detection of disintegrated neurons in the tissue slices of the CA1 area of the rat hippocampus. As expected in both control groups, we could not detect any FJC positive neurons . Unsurprp < 0.001), resp. 21-fold (p < 0.001) compared to both control groups (naïve and Met treated) and differences between naïve ischemized and Met ischemized groups represented 36.4% (p < 0.01). Remarkably, longer reperfusion time led to an apparent reduction of the cellular FJC positivity representing 32.3% (p < 0.001), resp. 44.6% (p < 0.001), when compared to the naïve IR-7d and IR-3d groups and Met-IR-3d group, probably as a result of an extensive tissue breakdown. This can be seen by the morphologically changed neurons (arrowhead), as well as scarred, unstained neuronal tissue which substitutes neuronal loss (asterisk), likely as a result of prolonged ischemia and neuronal tissue remodelling (In the prolonged reperfusion period (7 days) in both groups of animals (naïve and Met diet treated), we documented persisted increase of FJC+ neurons , NeuN antibody labelled nuclei as well as the cytoplasm of the majority of neuronal cell types of all regions in the adult brain . No immunoreactivity was observed in the astrocytes, neither in the nuclei nor the cytoplasm. The preponderance of the cytoplasmic immunopositivity was concentrated in the soma, extending to a short distance into the processes  decrease in the number of NeuN+ neurons in comparison to the naïve control. In the same group with the Met diet, we found a statistically significant decrease of NeuN+ neurons in comparison to the naive controls to 44.9% (p < 0.001) as well as to 51.4% (p < 0.001) compared to the Met controls (In the naïve ischemized group with 3 reperfusion days (IR-3d), we detected 36.7% (controls . Additiop < 0.001) in the naïve ischemized group, as well as in the ischemized Met group up to 49.2% (p < 0.001), when compared to the naïve controls and 43.1% versus Met controls (p < 0.001). In addition, Met diet induced diminishing in the number of maturate neurons between naïve and Met group up to 31.7% , in comparison to the naïve control (Immunolabelling of GFAP in the naïve controls (C) presented astrocytes with radially arranged thin processes. Astrocytes were dispersed in all layers of the hippocampus —arrow. I control . p < 0.01) when compared to the naïve controls. However, in the same Met ischemized group, surprisingly, we found a diminished number of astrocytes (p < 0.01) versus naïve control, and 34.4% (p < 0.001) versus Met-C . In addition, an extensive elevation was detected when compared to the 3 days reperfusion group, up to 28.9%  showed a significant decrease in tNAA/tCr ratio, and a significant increase in mIns/Cr when compared to the non-ischemized (C and Met-C) groups (Quantification of ) groups . However) groups .p = 0.031). The hippocampal volume was significantly increased in Met-IR-3d and significantly decreased in Met-IR-7d group, compared to the naïve control (C) as well as to the Met-C groups (p < 0.001) when compared to the 3rd post-ischemic day. With respect to the naïve control volume threshold level, we found an increased level in Met-C (10 ± 2%) and in the Met-IR-3d (23 ± 6%) animal groups, however, in the Met-IR-7d group (−6 ± 2%) was confirmed hippocampal volume decrement  or dysregulation in neurogenesis, as a reaction to the mild hHcy conditions . Based o1H MRS and MRI-volumetry analysis were applied as a non-invasive approach to determine the metabolic ratio and alternations in the hippocampal volume in ischemized animals with the Met diet. In vivo As generally accepted, tCr emerges as a constituent of all brain cells. tNAA is considered to be the characteristic neuroaxonal marker, tCho and mIns are regarded as the glial components, e.i. tCho (mainly for oligodendrocytes) and mIns for astrocytes ,52. 1H MRS revealed changes in all selected metabolites in the hippocampal area  can impair cardiac contractility, alter substrate metabolism, and cause cardiac remodelling and dysfunction . AnotherBased on these results, we suggest that Met-enriched diet inducing a mild form of hHcy in combination with IRI in rats represents an experimental model, which can evolve a toxic body environment with potentially detrimental impact to the neuronal tissue, seen on the morphological and metabolic levels. This is presented by the changed metabolic ratio, volume disturbances, attenuated neurites and activation of astrocytes in the rat hippocampus. In conclusion, our study utilizes a novel approach which combines histological analyses with the non-invasive in vivo 1H MRS, MRI methodology, with the aim to reveal the potential histo-morphological as well as the metabolic hippocampal dysregulation in rats with Met diet subjected to IRI. Both methods showed that hippocampi of ischemized animals with Met diet manifested exaggerated neurodegeneration, mainly in the selectively vulnerable CA 1 hippocampal region. Evidence of a possible causal link between the ischemic insult with hHcy induced by the Met diet and incipient neurodegenerative processes would contribute to better understanding of the potentially deleterious impact of both modifiable risk factors (hHcy and IRI) in clinically relevant neurological disorders. 1H MRI and histology Furthermore, there are different areas in the hippocampus with different vulnerability to acute or chronic insults that could influence the overall metabolic ratio. Our results support these findings and the need for a multiparametric MRI approach, in combination with histologic analysis to characterize the complex evolution of neurodegenerative diseases [The limitation of this study was in the measurement of metabolites because their level could fluctuate in chronic conditions due to the occurrence of cytotoxic oedema (tissue water), which could result in a lack of correlation between diseases .


This TQ delivery system was optimized by a three-factor, three-level Box-Behnken design. The optimized TQ-phytosome size was (45.59 ± 1.82 nm) and the vesicle size was confirmed by transmission electron microscopy. The in vitro release pattern of the formulation indicated a biphasic release pattern, where an initial burst release was observed within 2 h, followed by a prolonged release. A remarkable increase in dose-dependent cytotoxicity was evident from the significant decrease in IC50 value of TQ-phytosomes (4.31 ± 2.21 µM) against the A549 cell line. The differential effect of TQ-phytosomes in cell cycle analysis was observed, where cancer cells were accumulated on G2-M and pre-G1 phases. Furthermore, increased apoptotic induction and cell necrosis of TQ-phytosomes were revealed with the annexin V staining technique via activation of caspase-3. In reactive oxygen species (ROS) analysis, TQ-phytosomes acted to significantly increase ROS generation in A549 cells. In conclusion, the sustained release profile with significantly-improved anticancer potential could be obtained with TQ by this phytosomal nanocarrier platform.Thymoquinone (TQ), a natural polyphenol, has been associated with various pharmacological responses; however, low bioavailability of TQ limits its clinical application. Thus, a novel phytosomal delivery system of TQ-Phospholipon Nigella sativa L., used as folk medicine in many tropical countries [Lung cancer is among the most frequent and severe cancer types. The early stages of lung cancer typically do not exhibit signs or symptoms. Coughing, weight loss, shortness of breath, and chest pain are the most common symptoms ,2. Five-ountries . The blaountries . The nonountries . The triountries . TQ poteountries . Howeverountries . Alternaountries ,13,14. Aountries ,16, lipoountries , niosomeountries ,18. The ountries . Howeverountries . This coountries . Because® 90H (90% hydrogenated soy phosphatidylcholine), was gifted by Lipoid GmbH . The rest of the chemicals were of analytical grade.TQ (purity ≥98%), was procured from Sigma-Aldrich Inc. . The phospholipid, PhospholiponOptimization of TQ-phytosome formulations was achieved using three-factor Box-Behnken statistical quality by design technique . This design was used to evaluate the effect of independent formulation variables, such as TQ to phospholipid molar ratio (A), process temperature , and reflux time  on the dependent variable (response) and vesicle size  of the phytosome formulations. The design generated 17 runs with different combinations of the three independent variables at their low (−1), medium (0), and high (+1) levels for optimization of TQ-phytosome formulation as shown in The best-fitting model for the measured response was selected by computing and utilizing predicted and adjusted determination coefficients. Adequate precision was also computed to confirm the suitability of the model. Statistical analysis of measured response was performed using analysis of variance (ANOVA). Interaction between the investigated variables was studied from the generated contour plots. The optimization of process parameters to achieve minimum vesicle size was performed using the numerical method .® 90H  were placed in a 100 mL round bottom flask and refluxed with 20 mL dichloromethane. Based on the experimental design data, refluxing was performed at a specific temperature for specified time-period followed by evaporation to around 5 mL. TQ-phytosomes was obtained by lyophilizing the concentrate for 72 h. The developed TQ-phytosomes was stored at 4 °C until further use.Refluxing followed by the anti-solvent precipitation method was used to develop TQ-phytosomes . Based o® 90H. TQ, Phospholipon® 90H, and optimized TQ-phytosome spectra were measured in the range of 4000–400 cm−1 using an FTIR spectrophotometer .Fourier-transform infrared (FTIR) analysis was utilized to investigate the interaction between TQ and PhospholiponThe vesicle size of the TQ-phytosome formulation was measured by the dynamic light scattering (DLS) technique using Zetasizer Nano ZSP  after dispersing phytosomes with deionized water . MeasureThe shape and aggregation of the TQ-phytosomes vesicles were assessed using TEM  by spreading one drop of suspended phytosome samples on a carbon-coated grid. Negative staining of the sample was performed using 1% phosphotungstic acid followed by drying at room temperature for 15 min before visualization.In vitro release study of TQ from the free-TQ suspension (in 0.25% carboxymethyl cellulose) and TQ-phytosomes was performed following the dialysis bag method of Alhakamy et al.  in 0.2 Mv/v) fetal bovine serum in a humidified environment (5% carbon dioxide (CO2), and 95% air) at 37 °C for 48 h. While non-cancerous endothelial cells (EA.hy926) were cultured in Dulbecco’s modified Eagle’s medium. Then, cells were incubated with different concentrations of the optimized TQ-phytosome, blank-phytosomes, and free-TQ using a range of concentrations with reference to TQ at logarithmic intervals for 48 h at 37 °C in a CO2 incubator. A commercially-available MTT assay kit was used in determination of IC50 values based on the manufacturer’s instructions .The human lung cancer cell line (A549) was obtained using a cell strain from the American Type Cultural Collection (ATCC). The cell line was cultured as a monolayer in Roswell Park Memorial Institute (RPMI) 1640 cell culture media supplemented with penicillin (10 U/mL), streptomycin (10 µg/mL), amphotericin B (0.25 µg/mL), and 10% (5 cells) was seeded into a 96-well tissue culture plate followed by 24 h incubation in a 5% CO2 incubator at 37 °C for cell attachment. Optimized TQ-loaded phytosomes at concentrations ranging from 1 to 200 µM, blank-phytosomes, and free-TQ were added to a 96-well plate followed by 24 h incubation at standard conditions. Thiazolyl blue tetrazolium bromide (MTT) assay using a commercially-available kit  was performed to determine IC50 values of all the samples to study cell viability potential. The studies were performed in triplicate.The cytotoxicity of TQ-phytosomes was investigated against the A549 cell line. The cell suspension  was used for staining. Following incubation, the cells were centrifuged and re-suspended in 1× binding buffer (500 µL). Propidium iodide (BD Bioscience) and annexin V-FITC (5 μL of each) were added and the cells were incubated for 5 min at room temperature in the dark environment followed by flow cytometry analysis.Cell apoptosis was investigated using the reported dual staining technique . The cel4 numbers of A549 cells per well was incubated with 2.1 µM of the optimized TQ-phytosomes, blank phytosomes, and free-TQ in a 96-well plate. Additionally, a control group of cells without any treatment was kept in this study. The caspase-3 activity of the tested samples was evaluated by measuring the absorbance of the cell lysate at a wavelength of 405 nm.Caspase-3 activity was performed using a commercial kit (BD Biosciences) . The cel5 cells in each well of a 96-well plate was incubated with free-TQ, TQ-phytosomes, and blank-phytosomes for 24 h. A control group of cells without any treatment was also included. Further, 10 μM DCFH-DA was added and incubated for an additional half hour at 37 °C. Afterwards, the cells were washed with PBS and the fluorescence intensity was determined using a microplate reader at 485 nm (excitation wavelength) and of 530 nm (an emission wavelength).The ROS determination was carried out using A549 cells with a non-fluorescent probe, 2,7-diacetyl dichlorofluorescein diacetate (DCFH-DA) . The prot-test (two-tailed) and multiple t-test runs were carried out by means of one-way ANOVA followed by Turkey’s post hoc test. Statistical analyses were performed using IBM SPSS® statistical software . Outcomes were indicated as being statistically significant at p <  0.05.Replicates of three data for each sampling point were presented as mean ± SD. Data sets of two groups were analyzed using the The generated runs with different combinations of the three independent variables for optimization of the TQ-phytosome formulation are represented All the prepared TQ-phytosomes were nano-sized with vesicles size ranging from 43.7 to 129.9 nm. The observed and predicted values for the vesicle size are compiled in p-values in 2) were also significant at the same level.The statistical outcome for the vesicle size is presented in The equation relating the vesicle size to the independent variables according to the fitted model was generated by the software (Equation (1)). Furthermore, the effect of the variables on the vesicle size and the interaction between them are illustrated in the three-dimensional and contour plots presented in The numerically-optimized values for independent variables and the predicted responses obtained from Box-Behnken statistical design are displayed in The size and morphology of the optimized TQ-phytosomes were analyzed by TEM. The results obtained from the DLS method were in agreement with the vesicle size of the TEM micrograph . On furt−1, 1643 cm−1 due to the conjugated carbonyl group and 2965 cm−1 stretching bond of the dialiphatic CH2 group, while Phospholipon® 90H, showed sharp peaks at 2849 and 2923 cm−1 for the two amino group salts which confirmed at 1650 cm−1 and at 1740 cm−1 which represent the ketone group. Decreasing peak strength of the major groups in addition to disappearing of the Phospholipon® 90H peak (3017 cm−1) indicated the engagement of the function groups of TQ and Phospholipon® 90H together in the prepared phytosomes.As represented in The in vitro release pattern of TQ from the optimized TQ-phytosomes and free-TQ suspension is presented in 50 value was observed with TQ-phytosomes  and necrosis. Incubation of the A549 cells with the tested samples for 24 h, together with flow cytometry analysis and annexin-V/FITC and propidium iodide staining results are represented in Analysis results of caspase-3 using the commercial kit are represented in The induction of ROS using DCFH-DA by the fluorescein microscopy was measured in A549 cells following incubating with blank-phytosomes, free-TQ, and TQ-phytosomes . The cle2 value of 0.9935 and adjusted R2 value of 0.9962, in addition to adequate precision value of 61.26 (greater than 4) ensured the quadratic model validity. Furthermore, the observed and predicted values for the vesicle size, presented in Regarding the statistical analysis of vesicle size data, the good agreement between R50phospholipid complex within the architecture of the optimized phytosome, indicating that phytosomes act as an effective delivery system for TQ. The findings of value of the free-TQ against A549 cells are in agreement with the recent report by Acharya et al. [50 value of blank-phytosomes are similar to the published report by Alhakamy et al. [50 value of TQ when delivered by this phytosome nanocarrier successfully reduced the IC50 value by almost 45%, indicating the superiority of the delivery tool. This is similar to a previous study showing the cytotoxic activity of TQ in the human lung cancer cell line (A549 cells) [This initial burst release characteristics of TQ might be explained by the release of surface TQs from the phytosomes, because of easy dissociation of trapped TQs from the surface phospholipid of the phytosomes. Thereafter, there was a gradual release of TQ from the phytosomes with almost complete release within 12 h period, whereas it was around 49% from the suspension, indicating almost doubling of the amount of TQ released from the optimized phytosomes compared to the free drug suspension. Furthermore, the sustained release behavior of the TQ might be attributed to two different phenomena where initially the TQ was dissociated at the core of the stable phytosomes followed by diffusion of the dissociated TQ to the media through the phytosome skeleton . These fa et al. , whereasy et al. . The dec9 cells) . These c9 cells) ,37,38. H9 cells) . Moreove9 cells) ,27. Neve9 cells) ,16. This9 cells) . In rega9 cells) . These o9 cells) . In the 9 cells) ,38,39. I9 cells) . Our fin9 cells) .In summary, an optimized TQ-phytosome within the nanometer size range was developed using a Box-Behnken design tool. The spherical phytosomal deliveries were found to release the entrapped TQ in two phases, where the release was distinctly higher than that of the free-TQ suspension. Such improved performance of the TQ-phytosomes provided a 3-fold increase in the cytotoxic potential against A549 cells. Further, the differential role of TQ-phytosomes on A549 cell cycle analysis indicated the increased apoptotic potential of the nanocarrier, which was confirmed by the annexin V staining assay. This increased apoptosis of the cells was further established by the enhancement of caspase-3 expression which increased 2-fold in TQ-phytosomes in comparison with free-TQ treated cells. Additionally, ROS-based activation of Akt, leading to loss of mitochondrial membrane potential, might be another reason for increased apoptotic activity of the TQ–phytosomal treatment. Overall, this phytosomal delivery would be a promising nanocarrier cargo for delivering TQ; however, further extensive studies on in vitro and in vivo experimental models are necessary to establish this platform for effective treatment against lung cancer.


Endometrial carcinoma (EnCa) is one of the deadliest gynecological malignancies. The purpose of the current study was to develop an immune-related lncRNA prognostic signature for EnCa. In the current research, a series of systematic bioinformatics analyses were conducted to develop a novel immune-related lncRNA prognostic signature to predict disease-free survival (DFS) and response to immunotherapy and chemotherapy in EnCa. Based on the newly developed signature, immune status and mutational loading between high‑ and low‑risk groups were also compared. A novel 13-lncRNA signature associated with DFS of EnCa patients was ultimately developed using systematic bioinformatics analyses. The prognostic signature allowed us to distinguish samples with different risks with relatively high accuracy. In addition, univariate and multivariate Cox regression analyses confirmed that the signature was an independent factor for predicting DFS in EnCa. Moreover, a predictive nomogram combined with the risk signature and clinical stage was constructed to accurately predict 1-, 2-, 3-, and 5-year DFS of EnCa patients. Additionally, EnCa patients with different levels of risk had markedly different immune statuses and mutational loadings. Our findings indicate that the immune-related 13-lncRNA signature is a promising classifier for prognosis and response to immunotherapy and chemotherapy for EnCa. Endometrial carcinoma (EnCa), which originates from glandular epithelial cells of the endometrium, is the third most common type of malignant tumor in the female reproductive system worldwide Recently, it was proven that immunotherapies can be novel therapeutic strategies for EnCa and that the local immune status affects tumor progression and determines the response to immunotherapies In the current research, we developed a novel immune-related 13-lncRNA signature for EnCa patients that could accurately predict disease-free survival (DFS). In addition, based on the signature, the immune status and tumor mutation burden (TMB) in EnCa samples from different groups were distinctly varied. Notably, the findings obtained from this research may contribute to providing potential foundations for subsequent in-depth immune-related studies, to provide new insights into the individualized treatment of EnCa.https://cancergenome.nih.gov). DFS was defined as the time from the starting point to disease recurrence or death due to disease progression, which was treated as the end point of observation in this research. After integration of clinical information, a total of 481 samples were defined as an entire set, which was randomly divided into a training set and a testing set. The testing set with 240 samples and the entire set were used to verify findings obtained from the training set with 241 samples. Both the entire mRNA profile data and the clinical information of the samples are publicly available.Level-3 mRNA expression profiles and clinical data were collected from 552 EnCa and 23 normal samples in the TCGA database (http://www.broadinstitute.org/gsea/msigdb/index.jsp) http://www.gencodegenes. org) P < 0.0001).First, immune-related genes were obtained from the Molecular Signatures Database v4.0  and the testing cohort (n = 240). The training cohort was used for prognostic signature construction, while the testing cohort and entire cohort were used for validation of the signature. Univariate Cox regression analysis was applied to screen prognosis-related genes from 45 immune-related lncRNAs with the criteria of The risk score for each patient was then calculated using the following formula: risk score = -0.970896 * AL080317.2 + 0.173290 * ERICH6-AS1 - 1.091183 * AC016877.3 + 0.560300 * MCCC1-AS1 +0.153787 * AC120053.1 + 0.383809 * AC138932.5 - 0.690101 * ZNF433-AS1 - 0.123376 * SCARNA9 - 0.344099 * DBH-AS1 + 0.622167 * AL157932.1 + 0.236900 * AC073046.1 - 1.039016 * POC1B-AS1 + 0.428151 * AP003419.3. EnCa patients were divided into a high-risk group and a low-risk group at the cutoff of the median risk score. Univariate and multivariate Cox regression analysis was used to assess the prognostic values of risk score and other clinicopathological characteristics.http://cibersort.stanford.edu/) Transcriptome expression with standard annotation was uploaded to the CIBERSORT website (P < 0.0001). Then, the lncRNA-mRNA interaction network was visualized by Cytoscape software.We analyzed the potential coexpression relationships of prognostic lncRNAs by Pearson's correlation analysis  was performed to detect associated signaling pathways between the two groups Clinical stage and risk score, which had independent prognostic value, were incorporated to construct a nomogram to evaluate the probability of 1-, 2-, 3-, and 5-year DFS in EnCa. Validation of the nomogram was evaluated by calibration plot. The calibration curve of the nomogram was plotted to assess the nomogram-predicted probabilities against the actual rates.To compare the mutational loading between the two groups, the mutation annotation format (MAF) based on the TCGA cohort was functioned by the “maftools” package https://tcia.at/home).The immunophenoscore (IPS) of the tumor samples was calculated by analyzing the expression of genes in the following four major categories that determine immunogenicity: effector cells, immunosuppressive cells, MHC molecules, and immunomodulators In this study, the chemo-response to several drugs was predicted based on the online pharmaceutic database , this database performed a large-scale drug screen incorporating detailed genomic analyses to systematically identify drug response biomarkers. This database screened >1000 genetically characterised human cancer cell lines with a wide range of anti-cancer therapeutics. The sensitivity patterns of the cell lines are correlated with extensive genomic data to identify genetic features that are predictive of sensitivity. This large collection of cell lines enables this database to capture much of the genomic heterogeneity that underlies human cancer, and which appears to play a critical role in determining the variable response of patients to treatment with specific agents. The half-maximal inhibitory concentration (IC50) of each drug was detected in plenty of cell lines. Besides, we also used the pRRophetic R package. This R package established statistical models from gene expression and drug sensitivity data in a very large panel of cancer cell lines, then applying these models to gene expression data from primary tumor tissues. With the help of the data from GDSC and pRRophetic R package, we predicted the IC50 in patients. For individual patients in the TCGA group, the exact treatment scheme is different, in this study; we predicted the chemo-response of several drugs based on the genomic gene expression pattern in high-risk and low-risk groups. All these predictions were based on the online pharmaceutic database rather than the actual treatment response. All parameters were set by the default values with removal of the batch effect of “combat” and tissue type of “allSoldTumors”, and duplicate gene expression was summarized as the mean value P ≤ 0.05 was regarded as statistically significant.All statistical analyses were applied by R version 3.6.3 . For all analyses, two-tailed P < 0.0001).A total of 331 immune-related genes were collected from the Molecular Signatures Database. Then, 363 immune-related lncRNAs were identified according to Pearson correlation analysis  and entire sets (P<0.001)  Figs. C, 3C. Ad1) Figs. D, 3D. Fu1) Figs. A-F.P<0.05, P<0.05, P>0.05, A total of 13 hub lncRNAs were incorporated into the construction of the novel immune-related prognostic signature, so we next determined their expression and association with immune cell infiltration in EnCa. As shown in Immune-related mRNAs showing a Pearson correlation of > 0.5 with a 13-lncRNA signature were selected according to their association with lncRNAs, and altogether, 21 immune-related mRNAs were plotted into the network A-B.To define the potential biological processes associated with risk score in EnCa, GSEA was conducted to identify the enriched KEGG pathways. The results showed that “cell cycle”, “ECM receptor interaction”, “ERBB signaling pathway”, “TGF beta signaling pathway”, and “Wnt signaling pathway” were enriched in EnCa samples with high-risk scores A. Since P<0.05, P<0.05, Based on the newly developed signature, we next assessed the prognostic impact of the risk signature in EnCa patients with different clinicopathological characteristics in the entire cohort. First, patients who were older (aged > 60 years), had poor differentiation (G3&G4), and had the mixed and serous subtype tended to have higher risk scores P<0.05, A-C. MoreUnivariate and multivariate Cox regression analyses were used to evaluate whether the 13-lncRNA signature was an independent prognostic indicator for EnCa patients. In the univariate Cox regression analysis, the hazard ratios (HRs) of the risk score and 95% CIs were 1.189 (1.144-1.234), 1.060 (1.010-1.113), and 1.112 (1.083-1.141) in the training, testing, and entire sets, respectively  are shown in Fig. Moreover, to assess the correlations between risk score and tumor immune cell infiltration, CIBERSORT was applied to estimate the proportion of the 22 immune cells by using transcriptome data in each EnCa sample. We appraised that resting CD4+ memory T cells, M0 macrophages, and activated master cells were positively correlated with the 13-lncRNA risk score, while plasma cells, CD8+ T-cells, activated CD4+ memory T cells, regulatory T cells, activated dendritic cells, and resting master cells were negatively correlated with the 13-lncRNA risk score . The altered landscapes in EnCa with high- or low-risk scores are shown in Fig. P<0.05), while the other three scores had no significant association with the risk score , indicating that patients with high risk could be more resistant to these chemotherapies, which explains the poor prognosis of patients in the high-risk group  evaluations of patients with EnCa Among the 13 immune-related lncRNAs, ERICH6-AS1, MCCC1-AS1, AC120053.1, AC138932.5, ZNF433-AS1, DBH-AS1, AL157932.1, AC073046.1, POC1B-AS1, and AP003419.3 were risk-associated lncRNAs, while AC016877.3, AL080317.2, and SCARNA9 were protective lncRNAs. LncRNA-mRNA coexpression analysis was conducted to evaluate the functions of related lncRNAs. Several lncRNAs have been identified as functional genes in cancers. For example, DBH-AS1 may act as an oncogene, and downregulation of DBH-AS1 predicts better prognosis and suppresses osteosarcoma progression by inhibiting the PI3K/AKT pathway In recent years, immunotherapy has been considered a novel therapeutic strategy for EnCa As an important part of the current research, we evaluated the association between the expression of 13 hub lncRNAs and immune cell infiltration. Most lncRNAs were correlated with specific immune cell infiltration; among them, ERICH6-AS1 had the strongest correlation with immune cell infiltration. In addition, the risk model based on the expression of 13 immune-related lncRNAs was also significantly associated with different immune statuses in EnCa. TMB is an emerging biomarker for assessing the response to PD-1/PD-L1 inhibitors Although immunotherapy could be a novel therapeutic strategy, EnCa is usually treated with a combined regimen of surgery and chemotherapy However, the current research has several unavoidable limitations. Most important of all, the research is based on bioinformatics analysis, and there is no external EnCa data as a validation set; secondly, there were no recruited cohorts for verification of prognostic value of the signature in EnCa and response to immunotherapy and chemotherapy in high‑ and low‑risk groups. Whatever, we will pay more attention to emerging external data for further verifying our model in the future.In conclusion, we identified immune-related lncRNAs in EnCa and developed a 13-lncRNA signature that has significant prognostic value for patients with EnCa. Furthermore, high- and low-risk groups, which were divided based on the median risk score, displayed different immune statuses and responses to immunotherapy and chemotherapy.Supplementary figures and tables.Click here for additional data file.